International Journal of Systematic and Evolutionary Microbiology (2006), 56, 2089–2093 DOI 10.1099/ijs.0.

64040-0

Gracilibacter thermotolerans gen. nov., sp. nov., an

anaerobic, thermotolerant bacterium from a
constructed wetland receiving acid sulfate water
Yong-Jin Lee,1,2 Christopher S. Romanek,2,3 Gary L. Mills,2
Richard C. Davis,4 William B. Whitman1 and Juergen Wiegel1
1,3,4
Correspondence Departments of Microbiology1, Geology3 and Cellular Biology4, The University of Georgia,
Juergen Wiegel Athens, GA 30602, USA
jwiegel@uga.edu 2
Savannah River Ecology Laboratory, Aiken, SC 29802, USA

An obligatorily anaerobic, thermotolerant, asporogenic bacterium, strain JW/YJL-S1T, was isolated

from a sediment sample of a constructed wetland system receiving acid sulfate water
(pH 1?6–3?0). Cells of strain JW/YJL-S1T were straight to curved rods 0?2–0?4 mm in diameter and
2?0–7?0 mm in length, and stained Gram-negative. Growth of strain JW/YJL-S1T was observed at
25–54 6C (no growth at or below 20 or at or above 58 6C), with an optimum temperature range for
growth of 42?5–46?5 6C. The pH25 6C range for growth was 6?0–8?25 (no growth at or below
pH 5?7 or at or above pH 8?5), with optimum growth at pH 6?8–7?75. The salinity range for growth
was 0–1?5 % (w/v) NaCl, with an optimum at 0–0?5 %. During growth on glucose the isolate
produced acetate, lactate and ethanol as main fermentation end products. The fatty acid
composition was dominated by branched-chain compounds: i15 : 0, a15 : 0, i16 : 0 and i17 : 0. The
G+C content of the genomic DNA was 42?8 mol% (HPLC). Strain JW/YJL-S1T showed
polymorphism of the 16S rRNA gene. Its closest relative was the thermophilic Clostridium
thermosuccinogenes DSM 5807T (a member of Clostridium cluster III) (a BLASTN search
revealed Clostridium pascui DSM 10365T to have 92?7 % gene sequence similarity, the highest
value). The inferred phylogenetic trees placed strain JW/YJL-S1T between Clostridium clusters
I/II and III. Based on the morphological and phylogenetic data presented, JW/YJL-S1T (=DSM
17427T=ATCC BAA-1219T) is proposed as the type strain of a novel species in a new genus,
Gracilibacter thermotolerans gen. nov., sp. nov.

It is well known that various microbial communities are
involved not only in the generation but also in the
remediation of acid mine drainage. Most of the micro-
organisms isolated from mining environments are iron- and
sulfur-oxidizing bacteria and sulfate-reducing bacteria.
Although heterotrophic fermentative bacteria are closely
associated with other microbial communities in acid mine
drainage, little is known about their diversity and functions
in such environments. For instance, heterotrophic fermen-
tative bacteria can remove organic acids that can inhibit
chemolithotrophic bacteria such as Leptospirillum ferroox-
idans and Acidithiobacillus ferrooxidans (Johnson, 1998).
Therefore, the generation of acid mine drainage can be
facilitated by indigenous heterotrophic bacteria. Conversely,
they also support sulfate- or metal-reducing bacteria by
Abbreviation: PLFA, phospholipid fatty acid.
The GenBank/EMBL/DDBJ accession numbers for five clones of the
16S rRNA gene sequences of strain JW/YJL-S1T are DQ117465–
DQ117469.
degrading biopolymers into monomers and fermentation
products, which then serve as substrates, thus contributing
to the bioremediation of acid mine drainage.
Here we report on a new isolate recovered from a
constructed treatment wetland system receiving acid sulfate
water. On the basis of the physiological and phylogenetic
evidence presented, we propose a new genus, Gracilibacter,
to accommodate this organism.
Strain JW/YJL-S1T was isolated from an MPN (most
probable number) tube inoculated with sediment from
the upper layer of a constructed treatment wetland system.
This wetland was receiving water from an acid sulfate runoff
pond from a coal pile located at the Department of Energy’s
Savannah River Site near Aiken, SC, USA (Lee, 2005). The
acid runoff pond water has a pH of 1?6–3?0 and relatively
high sulfate and ferric iron concentrations. The uppermost
sediment in the constructed wetland was dominated by iron
oxyhydroxide precipitates coating and replacing an organic
substrate amended with limestone. The organic substrate

64040 G 2006 IUMS Printed in Great Britain 2089

Y.-J. Lee and others
used for the constructed treatment wetland was composed
primarily of composted stable wastes and spent brewing
grains mixed with local farmland soil from South Carolina
(Thomas, 2003; Lee, 2005). Thus, no defined habitat can be
given for the novel taxon described herein.
The isolate was routinely cultured in a carbonate-buffered
basal medium (Widdel & Bak, 1992) supplemented with
20 mM acetate and 0?1 mM ferric citrate at pH25 u C 6?8
(Wiegel, 1998) and 37 uC under anaerobic conditions
(100 % N2) using a modified Hungate technique
(Ljungdahl & Wiegel, 1986). Single colonies were obtained
from dilution rows in agar- (1?5 % w/v) shake roll-tubes.
To ensure that a culture was derived from a single cell, the
isolate was purified by an additional five rounds of single
colony isolation using the agar-shake roll-tube method
(Ljungdahl & Wiegel, 1986). Surface colonies of strain
JW/YJL-S1T appeared after 2–3 days and were less than
1 mm in diameter, circular to irregular, mostly translucent
and filamentous. Cell morphology was observed via light
microscopy (Olympus VANOX phase-contrast microscope)
and electron microscopy (JEOL100CX transmission elec-
tron microscope). Vegetative cells in liquid culture were
straight to curved, 0?2–0?4 mm in diameter and 2?0–7?0 mm
in length (Fig. 1). Cells were either single or formed chains.
Infrequently, cells up to 45 mm in length were detected. No
active motility was observed under phase-contrast micro-
scopy when cells were grown in carbonate-buffered basal
medium. However, retarded peritrichous flagella were
observed under the electron microscope (Fig. 1a; negative
staining). Motility was subsequently observed during
growth in SIM agar medium (Cappuccino & Sherman,
1987). No spores were detected either by microscopy or by
heat treatment (10 min at 80 uC). Gram staining was
performed according to standard procedures (Doetsch,
1981) and showed that cells from both the early exponential
and the stationary growth phases in both media stained
Gram-negative. Detailed results of morphological and
Fig. 1. Electron micrographs of cells of strain JW/YJL-S1T showing retarded flagella (a) and the prototypical image of the cell
line (b). The inset in (b) shows a thin section that matches a conventional thin section of a Gram-positive bacterium that has
an S layer. Bars, 2 mm (a), 4 mm (b) and 50 nm [inset in (b)].
2090
physiological characterization are given in the genus and
species descriptions below.
Using a temperature-gradient incubator (Scientific Indus-
tries, Inc.), the temperature range for growth was determined
to be 25–54 uC with an optimum at 42?5–46?5 uC. No growth
was detected at or below 20 uC or at or above 58 uC. The pH
range for growth was determined at 37 uC in basal medium
supplemented with 10 mM each of MES, HEPES and TAPS.
The pH25 u C range for growth was 6?0–8?25 with an
optimum at pH25 uC 6?8–7?75. No growth was detected at or
below pH 5?7 or at or above pH 8?5, suggesting that strain
JW/YJL-S1T originated not from the acid runoff but from the
organic substrate of the constructed wetland. The salinity
range for growth was 0–1?5 % (w/v) with an optimum at
0?5 % NaCl plus KCl at a ratio of 9 : 1; no growth was detected
above 2 % salts. The doubling time for strain JW/YJL-S1T was
3?1 h at 42 uC and pH25 uC 6?5 with 0?3 % yeast extract as the
substrate. The isolate required yeast extract for growth. For
characterization tests, cultures were incubated for up to
20 days, with growth judged positive if the optical density (at
600 nm) of the culture was twice that of a control culture
containing only yeast extract (0?02 %). Utilization of possible
substrates (0?2 %, w/v) was tested in the presence of 0?02 %
yeast extract. Strain JW/YJL-S1T used Casamino acids,
tryptone, peptone, maltose, sucrose, arabinose, fructose,
galactose, glucose, mannose, xylose, mannitol and sorbitol as
carbon and energy sources. No growth was observed with
cellobiose, lactose, raffinose, ribose, trehalose, inositol, xylitol,
acetate, lactate, pyruvate, methanol or carboxymethylcellu-
lose (1?0 % w/v, CMC 7LT or 7M; Hercules) as carbon and
energy sources. None of the following electron acceptors,
tested in media containing 20 mM lactate or 0?1 % yeast
extracts, was utilized: fumarate, nitrate, sulfate, sulfite,
thiosulfate, elemental sulfur, iron(III), anthraquinone 2,6-
disulfonate or manganese(IV) at concentrations of 20 mM
(sulfite was tested at 2 mM). Strain JW/YJL-S1T showed
positive growth on classical peptone-sugar media including
International Journal of Systematic and Evolutionary Microbiology 56

peptone-yeast extract (PY), peptone-yeast extract-glucose
(PYG), reinforced clostridial medium (RCM; Difco) and
thioglycolate broth (Difco). Fermentation end products from
20 mM glucose were analysed by HPLC with an Aminex-H87
column (Bio-Rad) and Beckman detector. The main organic
fermentation end products were acetate, lactate and ethanol.
Bacterial cell-membrane phospholipid fatty acids (PLFAs)
were extracted and isolated from lyophilized cells. Total
lipids were extracted, fractionated and saponified and then
methylated to obtain fatty acid methyl esters of the
phospholipids as described by Guckert et al. (1985). These
were then analysed by using a capillary column (30 m DB-5,
0?25 mm inner diameter) gas chromatograph (Agilent 6890)
equipped with a flame-ionization detector and by using a gas
chromatograph–mass spectrometer (Agilent 7972 MSD).
Compounds were identified by comparison of retention
times with a bacterial acid methyl ester standard (Supelco)
and by analysis of the mass spectra data. The PLFA
composition of strain JW/YJL-S1T was dominated by
branched-chain fatty acids (i15 : 0, a15 : 0, i16 : 0 and
i17 : 0), which accounted for 44?7 % of the total PLFAs.
Significant amounts of 16 : 0 (29?0 %) and small amounts of
the unsaturated 16 : 1, 17 : 1 and 18 : 1 fatty acids (5?4, 5?4
and 2?5 %, respectively) were also present (Table 1). This
profile is similar to that reported by O’Leary & Wilkinson
(1988) for closely related Gram-type positive bacteria. Anti-
biotic susceptibility was tested with ampicillin, chloramphe-
nicol, erythromycin, rifampicin, streptomycin and tetracy-
cline at concentrations of 10 and 100 mM. Strain JW/YJL-S1T
was resistant only to 10 mM streptomycin. Biochemical
properties of strain JW/YJL-S1T were determined using the
API ZYM system (bioMérieux). Enzyme assays of strain JW/
YJL-S1T were positive for esterase, leucine arylamidase, acid
phosphatase, naphthol-AS-BI-phosphohydrolase, b-galacto-
sidase, a-glucosidase and b-glucosidase. Strain JW/YJL-S1T
produced indole but not H2S in SIM medium.
For phylogenetic and G+C content analyses, DNA was
extracted by using a DNeasy genomic DNA purification kit
Table 1. PLFA composition of strain JW/YJL-S1T
Identified component Percentage of total
14 : 0 2?3
i15 : 0 23?8
a15 : 0 4?9
15 : 0 0?5
i16 : 0 0?6
16 : 1 5?4
16 : 0 29?0
17 : 1 5?4
i17 : 0 15?4
17 : 0 2?5
18 : 1v9c 1?6
18 : 1 0?8
18 : 0 3?5
http://ijs.sgmjournals.org
Gracilibacter thermotolerans gen. nov., sp. nov.
(Qiagen). The DNA G+C content was measured by HPLC
as described by Mesbah et al. (1989) with the modification
of Lee et al. (2005), using S1 nuclease (Invitrogen) and
0?3 M sodium acetate (pH 5?0). The G+C content of the
genomic DNA was 42?8 mol% (HPLC), the mean of four
replicate analyses. 16S rRNA gene sequence analysis of strain
JW/YJL-S1T was carried out three times (yielding the same
results) with a bacterial domain-specific primer set, 27
forward and 1492 reverse (Lane, 1991). PCR amplification
was performed using the Easy-A high-fidelity PCR cloning
enzyme (Stratagene) with 30 cycles of denaturation (94 uC,
30 s), annealing (58 uC, 30 s) and extension (72 uC, 1 min)
after initial denaturation at 94 uC for 3 min. Final extension
was for 7 min at 72 uC. To check for possible heterogeneity
of 16S rRNA genes, the PCR product of the 16S rRNA gene
of strain JW/YJL-S1T was cloned and transformed using
the TOPO TA cloning kit (Invitrogen). Plasmid DNA was
extracted using an Eppendorf FastPlasmid Mini kit
(Brinkmann), amplified as described above, purified using
the QIAquick PCR purification kit (Qiagen) and sequenced
by Macrogen Inc. (Seoul, Korea). Similarities among partial
sequences were determined using SEQUENCHER version
4.1.4 (Gene Codes). Retrieved 16S rRNA gene sequences
were analysed using BLAST and then aligned manually using
CLUSTAL X version 1.81 (Thompson et al., 1997) to create a
multiple sequence alignment. A phylogenetic tree (Fig. 2)
was inferred by the neighbour-joining method (Saitou &
Nei, 1987) using Jukes–Cantor distance corrections (Jukes
& Cantor, 1969), with the phylogenetic analysis package
PHYLIP version 3.6a2.1 (Felsenstein, 2001). Five clones of the
16S rRNA gene (derived from a single cell colony) were
sequenced and revealed the presence of polymorphism of
the 16S rRNA gene in strain JW/YJL-S1T. Four of the five
sequenced clones grouped together with more than 99 %
similarity. The other sequenced clone showed about 2 %
divergence from the rest. When these 16S rRNA gene
sequences, containing 1550 bp [approximately positions
2107 to 1450 according to the Escherichia coli numbering
scheme (GenBank accession no. X80725)], were compared
using a BLASTN search against sequences in GenBank, they
yielded the same correlations, i.e. that strain JW/YJL-S1T
was closely related to uncultured clones mostly obtained
from methanogenic environments and to consortia includ-
ing those from rice paddy-field microcosms (Chin et al.,
1999; Hengstmann et al., 1999; Erkel et al., 2005), an oil
reservoir (Grabowski et al., 2005), a uranium reduction
enrichment plant (GenBank accession numbers DQ125504
and DQ125852) and methanogenic fermenter cultures
degrading acetate (Shigematsu et al., 2003), propionate
or butyrate (GenBank accession numbers AB221361,
AB232817, AB248637, AB232818, AB248624 and
AB248638). Using BLAST search, Clostridium pascui DSM
10365T (Clostridium cluster I based on the classification of
Collins et al., 1994) had the most similar 16S rRNA gene
sequence among bacteria with validly published names,
with 93 % similarity over the first 160 bp, 92 % for 1012 bp
and 96 % for 54 bp. In an inferred phylogenetic tree,
strain JW/YJL-S1T was placed distantly between Collins’
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Y.-J. Lee and others
Clostridium cluster I/II and III (Fig. 2) with Clostridium
thermosuccinogenes as its closest neighbour. Note that in the
recent emended description of the genus Clostridium
(Wiegel et al., 2005), cluster II species such as Clostridium
proteolyticum fall within cluster I and were classified as
members of cluster I (the genus Clostridium sensu stricto),
whereas cluster III members represent a new family. In
addition to this phylogenetic evidence, strain JW/YJL-S1T
showed different physiological properties from members of
Clostridium clusters I and III and from Oxobacter pfennigii.
Whereas most Clostridium species and O. pfennigii form
endospores, strain JW/YJL-S1T showed no evidence of
endospore formation. In addition, strain JW/YJL-S1T was
not cellulolytic, distinguishing it from Clostridium thermo-
cellum and related cellulolytic species in cluster III; nor did
it produce succinic acid or grow at elevated temperatures.
Strain JW/YJL-S1T was also metabolically more versatile
than O. pfennigii. Furthermore, the DNA G+C content of
strain JW/YJL-S1T (42?8 mol%) is significantly higher than
that of related clostridial species. Based on the polyphasic
evidence provided here, JW/YJL-S1T is proposed as the type
strain of a novel species in a new genus, Gracilibacter
thermotolerans gen. nov., sp. nov., belonging to the order
Clostridiales (Garrity et al., 2004) but without assignment to
a family.
Description of Gracilibacter gen. nov.
Gracilibacter (Gra.ci.li.bac9ter. L. adj. gracilis slender; N.L.
masc. n. bacter equivalent of Gr. neut. n. baktron rod or staff;
N.L. masc. n. Gracilibacter slender rod, referring to its cell
shape).
A member of the low-G+C (about 43 mol%) Gram-
positive subphylum Bacillus–Clostridium. Specific habitat
unknown. Anaerobic chemo-organotrophs. No spores
observed. The type species is Gracilibacter thermotolerans.
2092
Fig. 2. Phylogenetic dendrogram based on
16S rRNA gene sequences showing the posi-
tion of strain JW/YJL-S1T (bold type) among
members of the family Clostridiaceae. The 16S
rRNA gene sequence data used correspond to
E. coli ATCC 11775T nucleotide positions
1–1499. The tree was constructed using the
neighbour-joining method with Jukes–Cantor
distance corrections. Numbers at nodes repre-
sent bootstrap percentages (1000 replicates).
Numbers in parentheses for strain JW/YJL-S1T
indicate numbering of different 16S rRNA
gene sequences. Bar, 5 nt substitutions per
100 nt.
Description of Gracilibacter thermotolerans
sp. nov.
Gracilibacter thermotolerans (ther.mo.to9le.rans. Gr. n.
thermê heat; L. pres. part. tolerans tolerating; N.L. part.
adj. thermotolerans heat-tolerating).
Cells are straight to curved rods, 0?2–0?4 mm in diameter
and 2?0–7?0 mm in length. Gram-type positive (Wiegel,
1981) but Gram staining negative at all growth phases.
Autoplasts (L-shaped cells) occur infrequently during the
late-stationary growth phase. Non-motile although retarded
flagella (1–5 per cell) are present. PLFA profile is dominated
by branched-chain fatty acids: i15 : 0, a15 : 0, i16 : 0 and
i17 : 0. Temperature range for growth is 25–54 uC (no
growth at and below 20 uC or at or above 58 uC), with an
optimum at 42?5–46?5 uC. The pH25 u C range for growth
is 6?0–8?25 (no growth at and below pH25 u C 5?7 or at or
above pH25 u C 8?5), with an optimum at 6?8–7?75. The
salinity range for growth is 0–1?5 % (w/v), with an optimum
at 0?5 %. In the presence of 0?02 % yeast extract, Casamino
acids, tryptone, peptone, maltose, sucrose, arabinose,
fructose, galactose, glucose, mannose, xylose, mannitol
and sorbitol serve as carbon and energy sources. The main
organic fermentation end products from glucose are
acetate, lactate and ethanol. No indication of growth on
H2/CO2 (80 : 20, v/v) or the use of iron(III), nitrate, thio-
sulfate, elemental sulfur, sulfate, sulfite, MnO2 or fumarate
as electron acceptors. Positive for esterase, leucine arylami-
dase, acid phosphatase, naphthol-AS-BI-phosphohydrolase,
b-galactosidase, a-glucosidase and b-glucosidase (API
ZYM). Indole is produced in SIM medium. Resistant to
streptomycin (10 mM). The G+C content of the genomic
DNA is 42?8 mol% (HPLC).
The type strain, JW/YJL-S1T (=DSM 17427T=ATCC BAA-
1219T), was isolated from a constructed treatment wetland
system at the Department of Energy’s Savannah River Site,
International Journal of Systematic and Evolutionary Microbiology 56

Aiken, SC, USA. The 16S rRNA gene of the type and so far
only strain exhibits the presence of polymorphism.
Acknowledgements
This research was partially supported by Financial Assistance Award
Number DE-FC09–96SR18546 between the United States Department
of Energy and the University of Georgia. We thank Robert C. Thomas
for providing samples for the experiments and Jean P. Euzéby for his
help with the nomenclature.
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